Discovery, engineering and application of a maleate isomerase for efficient L-malic acid biosynthesis via dual-enzyme cascade with fumarase.

L-malic acid (L-MA) is a valuable organic acid widely used in food, pharmaceuticals, and cosmetics. This study developed an efficient enzymatic system for its green synthesis using low-cost maleic acid as a substrate. Through bioinformatics mining, a highly active maleate isomerase from Schaalia vaccimaxillae (SvMI) was identified from candidate sequences, and its catalytic activity was further enhanced by 153.6% guided by computational predictions from existing deep learning-based tools, yielding the optimized mutant A124V. The mutant enzyme was co-expressed with fumarase BtFum in Escherichia coli to construct a one-pot dual-enzyme cascade system. Under optimized conditions (pH 8.0, CaCO₃ as neutralizer), the system efficiently converted 150 g/L maleic acid into 171.4 g/L L-MA with a productivity of 14.28 g·(L·h)-1, demonstrating high productivity and excellent industrial application potential. This work provides a sustainable and economically viable biocatalytic route for L-MA production.